ROLES OF VASCULARIZATION AND INNERVATION IN REGENERATIVE MEDICINE Skeletal muscle defects, such as those presented from traumatic injuries such as severe car crashes, cancer resections, or battlefield injuries, represent a significant healthcare problem. These large scale injuries overwhelm the innate repair mechanisms present in skeletal muscle and result in the clinical pathology termed volumetric muscle loss (VML). The current standard of care for VML repair is an autologous graft, which has a reduced functional outcome and is limited by re-innervation and re-vascularization, which may ultimately result in graft failure via tissue necrosis. There are several tissue engineered strategies designed to treat VML defects; however, none of these strategies simultaneously target vascularization and innervation. Tissue regeneration includes a complex set of coordinated events involving the growth, re-vascularization, and re-innervation of new tissue. Often, the success of tissue engineered constructs is limited by their ability to integrate with host vascular and neuronal tissue. The extent to which these systems communicate to support regeneration remains poorly understood. We hypothesize that vascularization and innervation are critical processes that are required to direct and sustain cell migration and differentiation in tissue regeneration. Further, we hypothesize that the signaling between vascularization and innervation are complementary to instruct regeneration. We will investigate the temporal nature of these signaling mechanisms to determine if vascularization precedes innervation, or vice versa, in mammalian regeneration by designing a biomaterial system where the distance between the two cell types and the availability of extracellular matrix molecules will be systematically varied to assess vascular and neuronal network formation (Aim 1). Concurrently, we will assess the ability of soluble factors within biomimetic constructs to model vascularization and innervation by determining the maturity and functionality of these tissue structures in a controlled in vitro environment (Aim 2). Finally, to address the clinical need of craniofacial VML injuries, we will develop a vascularized and innervated skeletal muscle model to understand how these processes affect and instruct skeletal muscle tissue formation by measuring force production of tissue constructs (Aim 3). The overall goal of this proposal is to generate an in vitro culture system to understand the interactions between vascularization and innervation processes, to elucidate signaling mechanisms involved, and ultimately to identify strategies to enhance tissue regeneration.